PTC bioinformatics - PimaBiotech135 - home

websterhissΒιοτεχνολογία

1 Οκτ 2013 (πριν από 3 χρόνια και 8 μήνες)

65 εμφανίσεις


1

The restriction enzyme HaeII cuts or cleaves DNA at the GGCC sequence. If the individual has
that sequence the restriction enzyme will cleave the gene at that locality. Non tasters do not show
this sequence and so in this 221 base pair region of the DNA, t
he segment stays whole. If a
classmate was a taster, their DNA would be cleaved leaving a 44 and 177 base pair segment,
which would be able to be seen through gel electrophoresis. What would be seen specifically in
gel electrophoresis is a single stand for

non tasters, and two split segments for those with the
ability to taste bitter (the PTC paper
)


Note that the actual SNP on either allele is either a G or a C. If a C was present at postion
145 on both alleles, the genotype is predicted to be TT,


If

a G
was present on both of the
student's alleles, they are designated a tt, for predicted nontaster. If a G was found on one
allele and a C was found on the other allele, the students indicate that they are
heterozygous, or Tt, or which is associated with a we
ak ability to taste PTC.


5726 TAS2R38
-

Taster

GCTTTGGGATAGATCTAGGCAAAGAGCTGGATGCTTTGTGAAGGAAAGGTCCTGGCT
TGGAACGTACATTTACCTTTCTGCACTGGGTGGCAACCAGGTCTTTAGATTAGCCAAC
TAGAGAAGAGAAGTAGAATAGCCAATTAGAGAAGTGACATCATGTTGACTCTAACT
CGCATCCGCACTGTGTCCTATGAAGTCAGGAGTACATT
TCTGTTCATTTCAGTCCTGG
AGTTTGCAGTGGGGTTTCTGACCAATG
CCTTCGTTTTCTTGGTGAATTTTTGGGATGT
AGTGAAGAGGC
C
GG
C
ACTGAGCAACAGTGATTGTGTGCTGCTGTGTCTCAGCATCAG
CCGGCTTTTCCTGCATGGACTGCTGTTCCTGAGTGCTATCCAGCTTACCCACTTCCAG
AAGTTGAGTGAACCACTGAACCACAGCTACCAAGCCATCATCATGCTATG
GATGATT
GCAAAC
CAAGCCAACCT
CTGGCTTGCTGCCTGCCTCAGCCTGCTTTACTGCTCCAAGC
TCATCCGTTTCTCTCACACCTTCCTGATCTGCTTGGCAAGCTGGGTCTCCAGGAAGAT
CTCCCAGATGCTCCTGGGTATTATTC



Primer Sequence:








5'
-
CCTTCGTTTTCTTGGTGAATTTTTGGGATGTAGTGAAGAGGCGG
-
3’









5'
-
AGGTTGGCTTGGTTTGCAAT
CATC
-
3'

Go to the
National Center for Biotechnology Information (NCBI) website,


http://www.ncbi.nlm.nih.gov/


1.

Select
BLAST on the top menu bar of the home page.

2.

Under Basic BLAST, clicked nucleot
ide BLAST.

3.

Past

the sequence of the primers into the search window (exc
luding "5'
-

and
-
3'").

Switch

the "Choose Search Set" dropdown menu to "Nucleotide collection (nr/nt)

Under program selection selecte

the radio button labeled "Somewhat similar sequence
s
(blastn)."

4.

Click ‘Blast


The information is split into three sections; Graphic Summary, Descriptions, and Alignments.


In the allignments section the primer sequence is aligned to the nucleotide sequence that was hit
in the search.


Notice that 43 of the

forward primer is missing, this is because the nucleotide did
not match the sequence.

Also In the Alignments section each alignment has an E
-
value The E
-
value is the number of alignments that would be expected by chance, the lower the E
-
value the
higher
the probability is that the hit is a close match to the query.


For example an E
-
value of 6e
-
12 means that this is a strong match from the database.


In the Descriptions section there are
many significant alignments that have E
-
values less than 0.1, they b
elong to humans and other

2

primates with the bitter taster receptor TAS2R38.


Also notice the sequence numbers on the
bottom of the seqeunces highlited by the green arrows this is where the primers have landed on
the subject sequence.


I
n the alignments sec
tion click

on the PTC Taster (
ref|NM_176817.3|
)


The top of the page contains information
about the sequence, such as basepair length, database
accession number, source, and references.


The middle of the report there are annotations of gene
and regulatory features.


The bottom section has the entire nucleotide sequence of the DNA
sequence that

contains the PCR product,


List the title of two studies linked to this nucleotide sequence


Return

to the NCBI home page and clicked on "
mapviewer
."


In the table find

Homo sapiens


and click

on the B icon next to the build with the highest number


Enter

the primer sequences into the search window, except for the non
-
nucleotide letters.


Selected BLASTN from the drop
-
down menu then clicked "Begin Search"


1.

Then clicked "View Report"

2.

Next clicked "Human genome view"

3.

Notice this sequence is located on chromo
some seven.




Click

on chromosome seven to go to the TAS2R38 locus,

The view can be adjusted to zoom out/in or move up/down the chromosomes to look at
neighboring genes.


The genes found on either side of TAS2R38 are CLECF5S (a lectin domain
gene) and MGA
M (a gene that encodes for a starch digesting enzyme).


These genes have
multiple coding exons while TAS2R38 has only one coding exon.


Most of the genes in this area
are mostly taste and olfactory receptors.


Go to NCBI Home page

In the dropdown search me
nu select ‘HomoloGene


What does this data base allow you to search?


Search the HomoloGene data base for the PTC Gene:
5726 TAS2R38


Scroll down to the protein Alignment section

Click on
Show Pairwise Alignment Scores


1.

List the percentage of similarity between the Human version of
5726 TAS2R38

and the
other mammals listed.

2.

To compare the Human and Chimpanzee protein sequences click on “
Blast” on the right
side of the data table.


3.

How many proteins differences are there between humans and chimpanzees?

4.

Repeat this same calculation for Humans and
Bos Taurus, domestic cow

5.

and Mus musculusm the common house mouse.


6.

Exp
lain why the this patter
n of similarity exits for these 3 mammals.